Cyclosporine is a potent immunosuppressive agent that selectively inhibits T cell ativity and is used to reduce the tissue rejection following organ transplantation. It is administered either orally or intravenously. The distribution of cyclosporin in blood is concentration dependent, but approximately 33-47% is in plasma, 4-9% in lymphocytes, 5-12% in granulocytes, 41-58% in erythrocytes. There are many factors that affect the cyclosporine absorption and metabolism, and there is poor correlation between the administered dose and the blood concentration, thus drug monitoring is essential to achieve optimal immunosuppressive effect in patients. In our laboratory we identified a patient that, after several weeks of aggressive dosing regimen of cyclosporine (1.3 mg/kg IV every 12 hours, whole blood cyclosporine 150 350 &#956;g/L) that was based on prior dosing history, suddenly had undetectable cyclosporine levels (i. e. a change from 188 to <25 &#956;g/L) when measured with the CEDIA Cyclosporine PLUS immunoassay (laboratory method). In contrast, the cyclosporine concentration for these specimens was 200 and 193 &#956;g/L when measured with HPLC-MS (reference laboratory). Review of the patients white blood count results showed that the falsely undetectable cyclosporine levels were associated with a highly abnormal granulocyte count (62.322 114.680 K/&#956;L, reference range 1.290 7.500 K/&#956;L) and normal mononuclear cells counts. To further investigate the effect of granulocytes on the immunoassay method whole blood specimens (EDTA) were spiked with cyclosporine dissolved in ethanol to a desired concentration and incubated at room temperature with gentle mixing for 10 minutes. The blood specimens were selected to have both normal (n=10) and abnormal (n=10) granulocyte counts. The spiked specimens were then treated with the lysing reagent and analyzed immediately. In addition, to assure complete lysis of all cells, aliquots of the lysed blood specimens were frozen prior to analysis. The cyclosporine recovery for specimens with normal granulocyte count was as expected (mean 104%). For specimens with increased granulocytes the recovery was between 60 and 88%, the lowest recovery associated with the highest count of 72.226 K/&#956;L. However, none of the specimens had undetectable (<25 &#956;g/L) cyclosporine levels. In addition, freezing of the specimens had minimal effect on the cyclosporine recovery. Based on these results we can suggest that granulocytes are completely lysed with the lysing reagent and the observed underestimation of cyclosporine in the presence of increased number of these cells is probably due to unidentified interferant of the assay reaction. Our laboratory noted a case in which a 24-hour urine specimen submitted for total protein analysis had a total protein concentration determined with pyrogallol red-molybdate method (LX20 analyzer, BeckmanCoulter, Brea CA) below the limit of detection (<60 mg/L), but dipstick analysis of the specimen indicated 2+ protein, i.e. approximately 1,000 mg/L (Aution 9EB strips on the Aution Max analyzer, Arkray, Kyoto, Japan). The concentration of urine albumin measured by the immunoturbidimetric method on the LX20 analyzer was 611 mg/L. After removal of low molecular weight components by ultrafiltration the total protein was 830 mg/L and albumin was 559 mg/L. These results indicated the presence of a low molecular weight component that interfered with the pyrogallol red-molybdate assay. The one unusual characteristic of this urine specimen was a strong dipstick reactivity for nitrite with no bacteria observed by microscopic examination. The nitrite reaction was positive (>0.001 mmol/L nitrite) up to a 300-fold urine dilution with 9% saline indicating a nitrite concentration of >0.3 mmol/L in the original urine specimen. The source of high urinary nitrite or other possible low molecular weight substances that reacted with nitrite reagent area of the dipstick was not identified. We investigated the effect of nitrite, nitrate and ascorbate on the pyrogallol red-molybdate and the biuret assays for total urine protein by spiking an albumin solution (850 mg/L, human fraction V, Sigma-Aldrich) in 0.9% saline with increasing amounts of sodium nitrite, sodium nitrate or sodium ascorbat to achieve their final concentrations of up to 10 mmol/L. Nitrite concentrations &#8805;1.0 mmol/L inhibited the pyrogallol red-molybdate assay while the biuret assay was not affected. The observed imprecision of the total protein measurements (7.6%) was in good agreement with the expected value of 7.5% CV. Nitrate and ascorbate that were run as controls had no effect on the pyrogallol red-molybdate assay. While the biuret assay was not affected by nitrate, ascorbate at the concentration of 10 mmol/L caused suppression of the measured protein concentration. Pyrogallol red-molybdate methods have become some of the most commonly used methods on automated analyzers. Pyrogallol red-molybdate methods react preferentially with polypeptides with high positive charge and to a lesser extent with proteins with a high negative charge, but reagents have been optimized to try to minimize differences in measurement of albumin and globulins. A number of compounds such as citrate, phosphate, tartrate, and oxalate cause a shift in the absorbance of the pyrogallol red-molybdate complex, but these effects can be suppressed by addition of excess oxalate to reagents. Results presented here suggest that nitrite is a potential negative interferent with protein measurements by pyrogallol red-molybdate methods. The concentrations of nitrite required to produce severe interference are quite high relative to usual physiological concentrations of nitrite. However, nitrite shows promise as a therapeutic agent; there are limited data about what concentrations of nitrite will occur in urine during nitrite therapy. The major diagnostic concerns from the interference of nitrite is the underestimation of urine protein excretion in the presence of a urinary tract infection by nitrate reducing bacteria or other exogenous sources of nitrite.